During the past decades the interest for preparation and commercialization of human milk oligosaccharides has been increasing steadily. The importance of human milk oligosaccharides is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities [1].
Sialylated human milk oligosaccharides such as disialyllacto-N-tetraose, 3′-O-sialyl-3-O-fucosyllactose, 6′-O-sialyllactose, 3′-O-sialyllactose, 6′-O-sialylated-lacto-N-neotetraose, 3′-O-sialylated-lacto-N-tetraose etc. are major components of human milk.
Among the above listed sialylated human milk oligosaccharides the sialic acid residue is always linked to the terminal 3′-O- and/or 6′-O— position(s) of D-galactose or to 6-O-positions of non terminal sugar residues via α-glycosidic linkages.
To date, access to large volumes of sialylated human milk oligosaccharides has not been possible by using isolation, biotechnology and synthetic methodologies. The chemical synthesis of sialylated human milk oligosaccharides is one of the most challenging fields of carbohydrate chemistry due to the nature of sialic acid donors themselves. In general, stereoselective glycosylations are achieved via neighbouring group participations but the lack of a substituent at C-3 position of sialic acid prevents such an option. Thus, stereoselective sialylation has to be achieved via careful selection of a sialic acid donor—acceptor match, kinetic and solvent effects. Furthermore, the presence of the carboxylic moiety at the anomeric position of sialic acid also creates unfavoured steric and electronic effects for stereoselective α-sialylations. The strong electron withdrawing effect of the carboxylic group initiates potential side reactions during the glycosylation via β-elimination.
Both the biological importance and the synthetic difficulties of sialylated oligosaccharides can be demonstrated via reviewing the background art of one of the simplest sialylated human milk oligosaccharides called 6′-O-sialyllactose (6′-SL, O—(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose, Scheme 1).

6′-O-Sialyllactose is one of the sialylated human milk oligosaccharides found in high concentration in mother's milk.
Several biological roles of 6′-O-sialyllactose such as its prebiotic, antibacterial, antiviral, immune system and cognitive development enhancing etc. effects [1] have been demonstrated. These important features make 6′-SL an attractive target for large scale production and product development for the nutritional and therapeutic industries. 6′-O-Sialyllactose has been synthesised by chemical [2], enzymatic [3] and biotechnological [4] methodologies or it has been isolated from natural sources [5]. However, these methodologies have not been attractive for scale-up due to lack of efficient purification methodologies, use of expensive and toxic reagents and the involvements of many synthetic steps.
Several chemical synthetic methods have been developed towards 6′-O-sialyllactose [2a-c], Na, K, Mg and Ca salts thereof [2d, e] or intermediates thereof [2f-j]. In summary, these strategies gives 6′-O-sialylated lactose via stereoselective 6′-O-sialylation of either 4′,6′-sugar diols or 6′-sugar alcohols using glycosylhalide, thioglycoside or diethylphosphite donor activations. The use of either very expensive or very toxic chemicals for the sialylation such as mercury cyanide, mercury bromide and silver carbonate is one of the reasons that make these methodologies less attractive for scale-up studies and production. Non efficient stereocontrol and/or yields likewise make(s) the strategies less suitable for large scale technology developments. Additionally, severe purification difficulties characterize all the listed synthetic strategies.
In case of enzymatic production of 6′-O-sialyllactose glycosyltransferases and sialidases are the preferred enzymes used [3]. These complex enzymatic systems represent very expensive methodologies for scale up productions of 6′-O-sialyllactose. Similarly, sialidases could not be used successfully in large scale production methodologies due to their lack of regio- and stereoselectivity. Low yields and difficult purification protocols are likewise a hindrance for industrial scale technology developments.
The isolation of 6′-SL from human and other mammals' milk is rather difficult even in milligram quantities due to the presence of a large number of similar oligosaccharides. To date, only analytical HPLC methodologies have been developed for the isolation of 6′-SL from natural sources [5].
Some biotechnological methodologies are also described using genetically modified bacteria, yeast or other micro organisms [4]. Such methodologies have serious drawbacks in regulatory processes due to limiting commercialisation opportunities.
The present invention represents the first commercial approach suitable for industrial manufacture of 6′-O-sialyllactose and other sialylated bioactive oligosaccharides. The successful strategy is based upon the introduction of novel sialic acid donor-acceptor pairs, novel sialic acid donors, novel diol-type lactose acceptors, relevant crystalline intermediates, the use of cheap and non-toxic activators and robust purification methodologies.